Skip to main content

Full text of "A Functional Dried Fruit Matrix Incorporated with Probiotic Strains: Lactobacillus Plantarum and Lactobacillus Kefir"

See other formats


www. f mf i-j ournal . org 



Focusing on Modern Food Industry (FMFI) Volume 2 Issue 3, August 2013 



A Functional Dried Fruit Matrix Incorporated 
with Probiotic Strains: Lactobacillus 
Plantarum and Lactobacillus Kefir 

Rego, A. 1 , Freixo, R. 2 , Silva, J. 3 , Gibbs, P. 4 , Morais, A.M.M.B 5 , Teixeira, P. 6 * 

CBQF - Centra de Biotecnologia e Quimica Fina - Laboratorio Associado, Escola Superior de Biotecnologia, 
Universidade Catolica Portuguesa/Porto, Rua Dr. Antonio Bernardino Almeida, 4200-072 Porto, Portugal 
^ngelamregoOhotmail.com; 2 ricardofreixol@hotmail.com; 3 jlsilva@porto.ucp.pt; 4 pgibbs@porto.ucp.pt; 
5 abmorais@porto.ucp.pt; * 6 pcteixeira@porto.ucp.pt 



Abstract 

The consumption of probiotic functional foods, i.e. processed 
foods enriched with microorganisms that confer health 
benefits to the host, shows a progressive increase in the last 
decade due to changes in habits and trends of consumers 
attracted by the benefits of these products. Currently, the 
development of fruits and vegetables with probiotic content 
is a topic of high interest for the probiotic-food consumers as 
these are a popular item perceived as healthy by consumers, 
and issues related with lactose intolerance are overcome. The 
aim of this research study was to develop a new healthy dry 
food that contains a source of probiotic strains providing 
some benefits to consumers. Apple was selected as an 
experimental food matrix and two different probiotic 
Lactobacillus species, L. plantarum and L. kefir, were tested 
separately. Samples were taken immediately before and after 
the drying process in order to determine the viability of 
bacteria adhered to the matrix. Dried apple cubes were 
stored in sterile closed glass containers or in sealed bags 
vacuum packed and normal atmosphere) at room 
temperature and at 4 9 C. The bacterial viability in the dried 
product was tested at different storage times. For both 
probiotic strains, a decrease of approximately 2 log cycles in 
bacterial cell numbers was observed after drying. The 
bacterial number in apple cubes at the time of storage at 
room temperature and 4 9 C was approximately 1x107 cfu/g. 
Both probiotic strains died after one month of storage at 
room temperature, while during storage at 4 Q C the cells 
remained viable after 3 months, with bacterial number 
around 1x106 cfu/g. 

Keywords 

Probiotic Bacteria; Storage; Survival; Tray Dryer; Immersion; 
Vacuum 

Introduction 

Fruits and vegetables are essential components of the 
human diet. Apart from being good sources of 
vitamins, minerals, and fibres, these foods are also a 



rich source of potentially bioactive compounds 
(Palafox et al., 2001). Additionally, the consumption of 
fresh fruits as well as functional foods e.g. probiotics, 
has increased considerably in recent years, due to the 
increasing concern in consumers about food and 
health. Combinations of fruits or vegetables with 
probiotics (Fito et al., 2001a; Alegre et al., 2010), would 
create a better, more convenient, product for 
consumers. 

Whilst dairy products are the priority of the 
development of novel probiotic foods (Puente et al., 
2009), lactose intolerance has been reported and an 
alternative to these dairy products is desirable. 

The incorporation of probiotic strains in several food 
matrixes has been studied maily due to their 
therapeutical benefits (Lourens-Hattingh et al., 2001; 
Shah et al., 2010). This represents a challenge, since the 
viability of the incorporated cells in the food matrix 
depends on several factors, such as pH, storage 
temperature, oxygen levels, and presence of 
competing microorganisms and inhibitors (Mattila- 
Sandholm et al., 2002). Vacuum impreganation has 
been reported by several authors as a technique to 
improve some food characteristics e.g. calcium, iron 
salts, pH depressors, antimicrobials, etc (Fito and 
Chiralt, 2000; Fito et al., 2000; Fito et al., 2001b) 

Probiotics can be added either to fresh foods with high 
water activity (aw) or to low aw dry foods. The fresh 
foods normally have a shelf-life of a few weeks, like 
yogurts, while the shelf-life of dried products is 
increased to months, as in the case of milk powder 
(Weinbreck et al., 2010). 

The principal objective of this research work was to 
create a new healthy dried (non-dairy) food containing 
a source of probiotic strains bringing some benefits to 



138 



Focusing on Modern Food Industry (FMFI) Volume 2 Issue 3, August 2013 



www.fmfi-journal.org 



consumers namely in the improvement of the immune 
system. Apple "Golden Delicious" was selected as the 
food matrix to perform the present research since it is i) 
easy to handle; ii) relatively cheap; iii) available in 
many countries at any time of the year; iv) highly 
nutritious (Obbagy et al., 2009) and v) has a highly 
porous matrix, allowing entry of the probiotic 
(Krokida et al., 1998). This apple variety is widely 
grown and available throughout the year 
(Anonymous., 2007). 

The specific objectives of the present work were: i) to 
establish the conditions for drying apple in a pilot 
scale tray dryer; ii) to evaluate the method for 
incorporating probiotic LAB (i.e. L. plantarum and L. 
kefir) in the apple matrix before the drying process, 
and iii) to evaluate the survival rate of the selected 
LAB during and after the drying process. 

Materials and Methods 

Probiotic Bacteria and Growth Conditions 

Two Lactobacillus strains, previously described as 
probiotic strains (Ouwehand et al., 2002; Vinderola et 
al., 2006; Golowczyc et al., 2007, 2009) were selected 
for the present study; L. plantarum, which can be found 
in fermented foods, and L. kefir, from kefir grains. 

These isolates belong to the culture collection of Escola 
Superior de Biotecnologia, Universidade Catolica 
Portuguesa. Both strains were stored in MRS broth 
(Pronadisa, Spain, Madrid) plus glycerol (30% v/v) at - 
80 2 C, until use. To prepare the pre-inoculum, a sterile 
tube with MRS broth (15 mL) was inoculated with a 
pure colony of the selected strain. This suspension was 
incubated at 37°C for 24 hours. The pre-inoculum (5 
mL, 1% v/v) was added to 500 mL of MRS broth and 
incubated at 37°C for 24 hours. This culture was 
centrifuged in sterile 50 mL Falcon tubes, (5000 x g at 
4°C; Hettich Zentrifugen Rotina 35R, Germany) for 5 
minutes followed by two washes of the pelleted cells 
by re-suspension and centrifugation, with sterile 
Ringer's solution (Merck, Germany, Darmstadt), under 
the same conditions. Cell pellets were then re- 
suspended in 20 mL of Ringer's solution in each 
Falcon tube in order to concentrate the probiotic 
suspension before addition to the fruit matrix. 

Sample Preparation 

The apples used in this study were the variety 'Golden 
Delicious' obtained in local markets from the region of 
Porto. This variety was chosen because of its sweet 



flavour, and the pulp is smooth with a crunchy texture 
(Molin, 2001) and as well it does not oxidize very 
easily during processing. Apples were washed with 
water, peeled and cut into cubes of about 2 cm sides, 
using a mold to obtain cubes with the same size and 
shape. These apple cubes were immediately immersed 
in sterile Ringer's solution to inhibit the oxidation of 
the matrix until submersion in the concentrated cell 
suspension prepared as described above (500 g of 
apple to 1L of sterile Ringer's solution). Cubes 
immersed in Ringer's solution were recovered by 
filtration using sterile gauze. 

Adherence of Probiotic Cells 

To promote adherence of probiotic cells into the apple 
matrix, two techniques were tested: immersion and 
vacuum impregnation (Betoret et al., 2003). 

1 ) Immersion 

In the immersion technique, the apple cubes were 
immersed in the probiotic suspension for one hour. 
In order to make this adherence uniform in all 
cubes, a gentle agitation over time was applied, 
ensuring that all cubes were immersed in the 
solution under the same conditions. Afterwards, 
the cubes were recovered by filtration under 
aseptic conditions using sterile gauze, placed on 
trays and then into the dryer (UOP8, Armfield, 
United Kingdom). 

2 ) Vacuum 

A vacuum impregnation technique was also tested 
(Maguina et al., 2002). Apple cubes were immersed 
in the concentrated cellular suspension, in a bag 
suitable for vacuum sealing. A pressure of 50 mbar 
for 1.2 seconds was established, with subsequent 
sealing of the bag (Multivac A300/52 Vacuum, 
United States of America). The bags were opened 
and the cubes were removed, also using sterile 
gauze, placed on trays that were loaded into the 
dryer, where the drying would be accomplished. 
Before and after addition to apple cubes, 
enumeration of LAB was performed as described 
below. 

Drying Conditions 

A pilot-scale tray dryer (which allows the drying of 
wet solid products by flowing hot air over the trays) 
was used. Two different temperatures and two 
different air velocities were tested. Initially, drying 
was performed at room temperature (ca. 20 Q C) and 



139 



www. f mf i-j ournal . org 



Focusing on Modern Food Industry (FMFI) Volume 2 Issue 3, August 2013 



with an average speed of air circulation of 0.5 m/s. 
These conditions were used to check if the probiotic 
bacteria, adhered to apple cubes by the immersion 
technique used for the sample preparation, suffered 
any decrease in viability, during dehydration. The 
duration of this experiment was one week and during 
this time several samples were taken and survivors 
enumerated. Subsequently, these conditions were 
changed. The temperature and speed of air flow were 
increased to 40 Q C and 1.5 m/s, respectively. In this 
second experiment, drying was faster essentially due 
to the increase in temperature. Drying of apple cubes 
with adhered LAB, by immersion at normal pressure 
or vacuum techniques, occurred in approximately, 27 
to 30 hours. Samples were taken during drying 
process and survivors were enumerated. The relative 
humidity (RH) of the drying air and the aw of the 
apple cubes during the drying process were measured 
in order to determine the effect of the RH on the 
drying of the cubes (Himmelfarb et al., 1962). 

Storage Conditions 

After drying at 40 Q C, the samples were divided into 
two groups, one to test the effect of atmospheric 
conditions, and the other to test the effect of 
temperature on the viability of the adhered probiotic 
bacteria. A portion of the dried apple was stored 
under vacuum conditions and the other in sterile 
Schott flasks (full flasks with almost no head space) 
under normal atmosphere conditions. Apple cubes 
that were submitted to vacuum conditions (Multivac 
A300/52 Vacuum, United States of America) were 
divided into bags and sealed after a pressure of 1 
mBar was established. These two groups of samples 
(vacuum packed and normal atmosphere) were stored 
and then divided in order to determine if storage 
temperature had any effect on cell viability. Two 
storage temperatures were tested: room temperature 
(ca. 20°C) and 4°C. 

Bacterial Enumeration 

One gram of apples (freshly inoculated) were added to 
9 mL of sterile Ringer's solution and mixed in the 
stomacher (BagMixer® 400 P, Interscience, France) for 
one minute. Then, serial decimal dilutions were 
performed and LAB were enumerated by the drop 
count technique on MRS agar (Biokar, France, 
Beauvais) plates. Colony counting was performed 
after incubation at 37 Q C for 24 hours. The same 
procedure was followed for samples after drying but 
the sample weight was 5 g added to 45 mL of sterile 



Ringer's solution. 
Results and Discussion 

The main objective of the present research was to 
create a dry fruit matrix with a high number of viable 
probiotic cells (at least > 1x107 cfu/g); therefore it was 
crucial: i) to obtain an initial suspension in which the 
matrix would be immersed, with a high cell 
concentration (-1010 cfu/mL); ii) to assure a high 
adherence of the probiotics to the fruit matrix; iii) to 
guarantee that after drying, the viability of adhered 
cells was still high. 

A concentrated probiotic suspension (ca. 1010 cfu/mL) 
was produced to allow a high incorporation of the 
cells into the food matrix by the two different 
techniques, immersion and vacuum. It was established 
that one hour of contact would be sufficient to 
promote good adherence with a high concentration of 
viable cells (ca. 109 cfu/mL; data not shown). 

In terms of cell numbers, after one hour immersion, a 
difference of one log cycle approximately, was 
observed between the initial probiotic suspension and 
the immersed apple matrix. According to Fito et al. 
(2001a), a vacuum technique would allow the 
introduction of controlled quantities of a solution into 
the porous structure of fruits. In fact, these authors 
described that vaccum impregnation could introduce 
into the fruit and vegetables, controlled quantities of a 
given solution. However, in the present study, no 
differences have been observed between the two 
tested techniques, since the same concentration of 
viable cells in the apple matrix was achieved at the 
same cell suspension concentration by both techniques 
(data not shown). 

Apple cubes with adhered probiotic bacteria were 
dried. After drying, it was observed that cubes that 
had been subjected to immersion under vacuum to 
promote adherence, presented lower viable numbers 
than apple cubes that had been subjected to immersion 
at normal pressure conditions (Figs. 1 and 2 for L. kefir 
and L. plantarum, respectively). In the course of 
comparison of both techniques, a reduction of 
approximately 2 log cycles was observed for both 
strains for apple samples that were just immersed, 
whereas losses were approximately of 4 log cycles for 
L. plantarum and near 3 log cycles for L. kefir for apple 
cubes that were vacuum treated. Therefore, the 
vacuum technique did not confer any advantages in 
either increasing the numbers of cells adhered or in 



140 



Focusing on Modern Food Industry (FMFI) Volume 2 Issue 3, August 2013 



www.fmfi-journal.org 



stability during drying. This was also confirmed in the 
study by Alzamora et al. (2005), also using apple cubes 
as a food matrix and a different range of vacuum 
pressures. 




-i 

FIG. 1. VIABILITY OF LACTOBACILLUS KEFIR CELLS IN APPLE 
CUBES AFTER ADHESION; ""^BY THE IMMERSION 
TECHNIQUE: BY THE VACUUM TECHNIQUE. ERROR 

BARS INDICATE NO VARIABILITY BETWEEN ASSAYS. 



Drying Time (hours) 




-5 



FIG.2. VIABILITY OF LACTOBACILLUS PLANT ARUM CELLS IN 
APPLE CUBES AFTER ADHESION; 1 BY THE IMMERSION 
TECHNIQUE; BY THE VACUUM TECHNIQUE. ERROR 

BARS INDICATE NO VARIABILITY BETWEEN ASSAYS. 

It was also noted that vacuum immersed samples had 
a worse visual aspect after drying, with more damage 
observed when compared with samples that were not 
subjected to vacuum. 

Even with the reduction of viable cells (<2 log cfu/g) 
observed in the apple cubes inoculated by simple 
immersion, the lactobacilli continued to be present in 
large numbers, (ca. 107 cfu/g), even at the end of the 
drying process. Much greater reductions of viable cells 
(ca. 3-4 log cfu/g) were noted for both lactobacilli in 
apple cubes inoculated by vacuum immersion (Figs. 1 
and 2). 

When these results were compared with Betoret et al. 
(2003), some differences in the final concentrations of 
incorporated cells in the final product were observed. 
To promote adherence (Betoret et al., 2003), fruit juices 
or even milk inoculated with probiotic bacteria were 
used. These suspensions were then put in contact with 



the apple slices to promote the incorporation of the 
bacteria in the matrix. This step led to a better 
incorporation of the probiotic bacteria into the apple 
slices, when compared to the results obtained in the 
current study. The use of juice or milk seemed to 
confer some protection to the probiotic cells, making 
them more resistant to drying. The pressure used for 
vacuum immersion was the same, but Betoret et al. 
(2003) applied it for a longer period. This may have 
had some advantages for the incorporation of the cells, 
since in the currently reported study, vacuum was 
applied for only 1.2 seconds at 50 mBar instead the 10 
minutes used by Betoret et al. (2003) to promote 
adherence. These differences in time could lead to 
different adherences of the cells to the matrix. 

Dried apple cubes incorporated with the two probiotic 
LAB by both methods, were stored at room 
temperature (ca. 20°C) in closed glass bottles in the 
dark for up to 25 days. After 24 days of storage, viable 
cells of L. kefir, incorporated by either method, had 
decreased by ca. 1 log cfu/g (Fig.3). 

Drying Time (hours) 














-0,5 ' 




-1 - 




-1,5 - 




-2 - 


1 




BC 




-2,5 - 


-! 






-3 - 




-3,5 - 




-4 - 




-4,5 - 



FIG.3. VIABILITY OF LACTOBACILLUS PLANTARUM CELLS IN 
APPLE CUBES DURING DRYING TIME;. ™^ IMMERSION 
TECHNIQUE TO PROMOTE ADHERENCE; - VACUUM 
TECHNIQUE TO PROMOTE ADHERENCE. ERROR BARS 
INDICATE NO VARIABILITY BETWEEN ASSAYS. 

After 20 days of storage, L. plantarum viable cells 
incorporated into apple by immersion, had decreased 
by ca. 0.5 log cfu/g, but cells incorporated by the 
vacuum technique had decreased by ca. 1.5 log cfu/g 
(Fig.4). In an attempt to minimize the loss of viability 
of L. plantarum, vacuum infused apple cubes were also 
stored under vacuum, since it was possible that air - 
oxygen — was deleterious for cell survival. After just 
eight days of storage at ambient temperature, vacuum- 
stored cells had lost ca. 4 log cfu/g, and could not be 
recovered thereafter; cells could be recovered after 25 
days storage in normal atmosphere, although with a 



141 



www. f mf i-j ournal . org 



Focusing on Modern Food Industry (FMFI) Volume 2 Issue 3, August 2013 



loss of viability of ca. 2 log cfu/g (Fig. 5). 



i 

().- 



g 

^05 



-1 
-1,5 
-2 



Storage time (Days) 



10 15 



25 30 



FIG.4. VIABILITY OF LACTOBACILLUS KEFIR CELLS IN APPLE 
CUBES DURING DRYING TIME;. ™^ IMMERSION 
TECHNIQUE TO PROMOTE ADHERENCE; VACUUM 
TECHNIQUE TO PROMOTE ADHERENCE. ERROR BARS 
INDICATE NO VARIABILITY BETWEEN ASSAYS. 



Sloraje lime (Days) 




FIG.5. EFFECT OF VACUUM TECHNIQUE USED TO PROMOTE 
ADHESION OF LACTOBACILLUS PLANT ARUM, ON ITS 
SURVIVAL DURING STORAGE AT ROOM TEMPERATURE. 
™^ STORAGE AT NORMAL ATMOSPHERE; STORAGE 
UNDER VACUUM. 

Over several replicated experiments with both LAB 
incorporated into apple cubes by immersion, and 
drying by air at 40°C, the cell viability losses by drying, 
were between 1.5 and ca 3 log cfu/g (data not shown). 
A possible reason for the differences recorded is that 
the RH of the heated air was not controlled, thus 
giving different rates of drying, and with low RH 
there may have been rapid surface dehydration and 
prolonged dehydration of the interior. 

Golowczyc et al., (2009) observed, using the same 
strains used in this study, that L. plantarum was more 
resistant to high temperatures than L. kefir. As 
reported by other authors, these strains of Lactobacillus 
are capable of growth in this range of temperature (De 
Vos, 2009), so cell death is probably the decrease in 
water content, leading to shrinkage of the cell 
membrane and to the death of cells. 

After one month of storage at room temperature, the 
viability of cells in apple slices decreased by 4 log 
cycles. However, storage at 4°C resulted in a loss of 



viability of only 1 log cfu/g even after 65 days of 
storage (Fig. 6). As in the study of Alzamora et. al. 
(2005), samples that were incorporated with probiotics, 
only lost one log cycle of viability during storage at 4 
Q C and could remain stable for long periods at that 
temperature. So, it was possible to conclude that, for 
the conditions investigated, storage in normal 
atmosphere at 4 Q C was the best way to preserve 
probiotic cell viability in dried apple cubes. 



Storage Ti me (Days} 




FIG.6. SURVIVAL OF CELLS IN DRIED APPLE CUBES DURING 
STORAGE. ""^ LACTOBACILLUS PLANTARUM SURVIVAL 
AT ROOM TEMPERATURE; LACTOBACILLUS 
PLANTARUM SURVIVAL AT 4 °C; LACTOBACILLUS 
KEFIR SURVIVAL AT ROOM TEMPERATURE; 
LACTOBACILLUS KEFIR SURVIVAL AT 4 Q C. 

After drying the apple matrix, the cubes were stored 
for at least one month, to check the cell viability and 
shelf life under storage conditions. Several factors 
could influence the quality of the product, including 
temperature, moisture content, and atmosphere 
composition in which the product is stored 
(Anonymous, 2001). 

ACKNOWLEDGMENT 

This work was supported by National Funds from 
FCT - Fundacao para a Ciencia e a Tecnologia through 
project PEst-OE/EQB/LA0016/2011. Financial support 
for author Joana Silva was provided by Postdoctoral 
fellowship SFRH/BPD/35392/2007 (FCT) 

REFERENCES 

Anonymous. Maca. Ministerio da Agriculture, do 

Desenvolvimento Rural e das Pescas, Gabinete de 

Planeamento e Politicas 2007, pp. 1-17. 
Anonymous, FDA. Evaluation and Definition of Potentially 

Hazardous Foods. U.S. Department of Health & human 

Services, FDA, 2001. 
Alegre, I., Vinas, I., Usall, J., Anguera, M., and Abadias, M. 

Microbiological and physicochemical quality of fresh-cut 



142 



Focusing on Modern Food Industry (FMFI) Volume 2 Issue 3, August 2013 



www.fmfi-journal.org 



apple enriched with the probiotic strain Lactobacillus 
rhamnosus GG. Food Microbiology 28 (2010): 59-66. 

Alzamora, S.M. et al., Novel functional foods from vegetable 
matrices impregnated with biologically active 
compounds. Journal of Food Engineering 67 (2005): 205- 
214. 

Betoret, N. et al., Development of probiotic-enriched dried 
fruits by vacuum impregnation. Journal of Food 
Engineering 56 (2003): 273-277. 

De Vos et al., Order II. Lactobacillales ord. nov. In: Bergey's 
Manual of Systematic Bacteriology: The firmicutes 
(Second Edition) pp. 464-510, 2009. 

Fito, P. et al., Vacuum impregnation and osmotic 
dehydration in matrix engineering Application in 
functional fresh food development. Journal of Food 
Engineering 49 (2001a), 175-183. 

Fito, P. et al., Vacuum impregnation for development of new 
dehydrated products. Journal of Food Engineering 49 
(2001b), 297-302. 

Fito, P. and Chiralt, A. Vacuum impregnation of plant 
tissues. In Alzamora, S.M. et al. (Eds.), Minimal 
processed fruits and vegetables (pp. 189-201). Maryland: 
Aspen Publishers (2000). 

Golowczyc, M.A., Silva, J., Abraham, A.G., De Antoni, G.L. 
and Teixeira, P. Preservation of probiotic strains isolated 
from kefir by spray drying. Letters in Applied 
Microbiology 50 (2009): 7-12. 

Golowczyc, M.A.; Mobili, P.; Garrote, G.L.; Abraham, A.G. 
and Antoni, A.G. Protective action of Lactobacillus kefir 
carrying S-layer protein against Salmonella enterica 
serovar Enteritidis. International Journal of Food 
Microbiology 118 (2007): 264-273. 

Himmelfarb, P., El-Bisi, H.M., Read,R.B. and Litsky,W. Effect 
of relative humidity on the bactericidal activity of 
propylene oxide vapor. Institute of Agricultural and 
Industrial Microbiology, University of Massachusetts 10 
(1962): 431-435. 

Krokida, M.K., Karathanos, V.T. and Maroulis, Z.B. Effect of 
freeze-drying conditions on shrinkage and porosity of 
dehydrated agricultural products. Journal of Food 
Engineering 35 (1998): 369-380. 



Lourens-Hattingh, A. and Viljoen, B.C. Yogurt as probiotic 

carrier food. International Dairy Journal 11 (2001): 1-17. 
Maguina, G. et al, Incoporation of Bifidobacterium spp by 

hydrodynamic mechanism in a porous fruit matrix. 

Annual Meeting and Food Expo - Anaheim, California, 

2002. 

Mattila-Sandholm et al., Technological challenges for future 
probiotic foods. International Dairy Journal 12 (2002): 
173-182. 

Molin, G. Probiotics in foods not containing milk or milk 
constituents, with special reference to Lactobacillus 
plantarum 299v. American Journal of Clinical Nutrition 
73 (2001):380 - 385. 

Obbagy, J.E. and Rolls, B.J. The effect of fruit in different 
forms on energy intake and satiety at a meal. Appetite 52 
(2009): 416-422. 

Ouwehand, A.C.; Salminen, S. and Isolauri, E. Probiotics: an 
overview of beneficial effects. Antonie van Leeuwenhoek 
82 (2002): 279-289. 

Palafox, H.C., Zavala, J. and Gonzalez, G.A. The role of 
dietary fiber in the bioaccessibility and bioavailability of 
fruit and vegetable antioxidants. Journal of Food Science 
76 (2011): 6-15. 

Puente, L.D., Betoret, N.V., Cortes, M.R. Evolution of 
probiotic content and color of apples impregnated with 
lactic acid bacteria. Vitae, Revista de la Facultad de 
Quimica Farmaceutica 16 (2009): 297-303. 

Shah, N.P., Ding, W.K., Fallourd, M.J. and Leyer, G. 
Improving the stability of probiotic bacteria in model 
fruit juices using vitamins and antioxidants. Journal of 
Food Science 75 (2010): 278-282. 

Vinderola, G., Perdigon, G., Duarte, J., Farnworth, E. and 
Matar, C. Effects of the oral administration of the 
exopolysaccharide produced by Lactobacillus 
kefir anofaciens on the gut mucosal immunity. Cytokine 36 
(2006): 254-260. 

Weinbreck, F., Bodnar, I., Marco, M.L. Can encapsulation 
lengthen the shelf-life of probiotic bacteria in dry 
products? International Journal of Food Microbiology 
136 (2010): 364-367. 



143